Background

Mesenchymal Stem Cells (MSCs) are a promising therapeutic tool in veterinary medicine. Currently the subcutaneous adipose tissue is the leading source of MSCs in dogs. MSCs derived from distinct fat depots have shown dissimilarities in their accessibility and therapeutic potential. The aims of our work were to determine the suitability of omental adipose tissue as a source of MSCs, according to sampling success, cell yield and paracrine properties of isolated cells, and compared to subcutaneous adipose tissue.

Conclusion

Compared to subcutaneous adipose tissue, omental adipose tissue is a more suitable source of MSCs in dogs. Since it can be procured from donors with any body condition, its collection procedure is always feasible, its cell yield is high and the MSCs isolated from it have desirable differentiation and paracrine potentials.

Mesenchymal stem cells (MSCs) are non-hematopoietic precursor cells that can be differentiated, among others, into chondrocytes, osteocytes and adipocytes [1]. Together, MSCs secrete trophic, vasculogenic and immunomodulatory factors that have a paracrine effect on tissue resident cells [2–4]. Hence, MSCs are a promissory therapeutic tool for regenerative medicine [5].

The leading source of MSCs is the bone marrow. In 2001, they were isolated for the first time from adipose tissue [6]. When compared to bone marrow, adipose tissue appeared as a superior source of MSCs due to the fact that a less invasive procedure is required to procure it [7–9].

In veterinary medicine, it has been proven that the administration of adipose-derived MSCs have therapeutic effects in small animal patients, particularly in cats and dogs [10–13]. Adult dogs have adipose tissue locates mainly in subcutaneous and visceral depots. Thus, adipose tissue may be procured through minimally invasive procedures from arms, thighs and abdomen (subcutaneous), or from omentum, kidney and liver (visceral) [14, 15]. While the abundance of subcutaneous adipose tissue depends on the body condition, the extent of omentum is relatively constant [16, 17]. It has been demonstrated that there are MSCs in the omentum of both dogs [10, 18] and humans [19, 20]. Omental-derived MSCs are similar to subcutaneous-derived MSCs according to their proliferation and surface antigen expression [18]. Up to our knowledge, no data are available regarding the abundance and paracrine potential of omental-derived MSCs. Since previous studies showed that adipose-derived MSC properties vary from depot to depot [21], it will be also interesting to compare MSCs isolated form omentum with those isolated from subcutaneous fat, the leading adipose source of MSCs.

The aims of our work were to determine the suitability of omental adipose tissue as a source of MSCs, according to sampling success, cell yield and paracrine properties of isolated cells, and compared to subcutaneous adipose tissue.

Fibroblast-like Colony forming unit (CFU-F) assay

CFU-F assay was performed on freshly isolated cells as previously described [24]. Briefly, 500 mononuclear cells/cm2 were cultured in expansion medium. At day 7, cells were fixed with 4% paraformaldehyde for 10 min and stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO) in 10% methanol for 20 min. Plates were observed under light microscope (Leica DM2000). Clusters containing more than 50 cells were scored as CFU-Fs and counted. Results were expressed as CFU-F per gram of tissue (CFU-F/g tissue).

Assays were performed in triplicate.

Evaluation of cumulative population doubling level (CPDL) and senescence

One thousand cells/cm2 were seeded and cultured with expansion medium. The medium was changed every three days and cells were subcultured when reaching 80% confluence. The population doubling (PD) at each subculture was calculated according to the formula PD = ln (Nf/Ni) /ln 2, where Ni and Nf are initial and final cell numbers, respectively. The PDs of continuous subcultures were added to obtain CPDL [10].

Senescence was assessed looking for changes in cell morphology such as cell enlargement, accumulation of vacuoles and presence of cellular debris [25].

Proliferation assay

Human fibroblasts were seeded at 4000 cells/cm2 and cultivated with alpha-MEM (control) or alpha-MEM conditioned by MSCs for 24 h. The medium was changed every 3 days. Three, six, nine and 12 days later, cells were stained with 0.5% crystal violet in 10% methanol for 20 min. After four washes, crystal violet incorporated into the cells were solubilized with 50% methanol in PBS and quantified spectrophotometrically (absorbance at 570 nm) [24].

Assays were performed in triplicate.

Scratch assay

Human fibroblasts were seeded at 8000 cells/cm2. After 24 h, a line in the monolayer was performed with a sterile p200 pipette tip and medium was changed by alpha-MEM (control) or alpha-MEM conditioned by MSCs for 24 h. Zero, six, and 12 h after scratching images were captured under a light microscope (Leica DM2000) with a digital camera (Leica DFC 295). Image J software (http://rsbweb.nih.gov/ij/) was used to quantify the scratch area [27].

Statistical analysis

Data are presented as mean ± S.E.M. To determine the statistical significance of intergroup differences a one-way ANOVA test was used to compare mean values among all groups and Student’s unpaired t-test or Mann-Whitney test (non parametric) was used to compare mean values between two groups. p < 0.05 was considered as statistically significant.

The age and weight of donor dogs were 10 ± 3 months and 12 ± 6 Kg (Table 1). Omental samples were procured from the 14 donors and subcutaneous samples were procured from 10 of them. Thus, the success sampling rates were 100% (14 of 14) for omental adipose tissue and 71% (10 of 14) for subcutaneous adipose tissue (Table 1). The average weight of the procured samples was 5.2 ± 4.5 g for omental and 2.4 ± 3.4 g for subcutaneal adipose tissue (Table 1).

Table 1

Enrolled animals and procured samples characteristics

Donor (identifier)

Age (months)

Weight (kilograms)

Subcutaneous (grams)

Omental (grams)

B001

8

8

not available

6.3

P002

12

15

4.8

8.1

L003

7

11

4.6

9.8

P004

8

12

not available

3.1

K005

12

15

1.9

2.3

L006

7

12

4.5

8.4

P007

8

10

3.2

4.5

C008

12

16

3.1

7.1

C009

11

6

not available

3.2

S010

8

15

5.8

5.9

O011

8

7

not available

3.7

C012

12

10

1.5

3.0

P013

10

6

2.3

4.8

S014

10

18

3.3

3.2

TOTAL

10 ± 3

12 ± 6

2.4 ± 3.4

5.2 ± 4.5

Further characterization of adipose-derived MSCs was performed for samples obtained from the 10 donors in whom it was possible to obtain both omental and subcutaneous adipose tissues.

Omental adipose tissue and subcutaneous adipose tissue have MSCs

Cells isolated from both sources adhered to plastic and showed fibroblast-like morphology (Fig. 1a). Together, they were negative for hematopoietic markers (CD45 and CD11b) and positive for MSC markers (CD90 and CD44) (Fig. 1b). When exposed to adipogenic stimulus, cells differentiated into adipocytes that accumulate lipid droplets throughout the cytoplasm as confirmed by Oil Red O staining (Fig. 1c). After 21 days under chondrogenic induction, sulfated glycosaminoglycans were present in the matrix as revealed by Safranin O staining (Fig. 1c). Cell osteogenic differentiation was confirmed due to the appearance, 3 weeks after exposure to osteogenic medium, of calcium deposits that stained with Alizarin Red (Fig. 1c).

MSCs derived from omental and subcutaneous adipose tissues have a similar expansion potential

No statistical difference was observed bewteen omental- and subcutaneous-derived MSCs regarding their proliferation potential up to passage 12 (73 ± 1 vs. 74 ± 1 CDPL) (Fig. 2c). Senescence characteristics such as cell enlargement, generation of vacuoles and presence of cellular debris were seen from passage 10 in both MSCs (Fig. 2d).

MSCs derived from omental and subcutaneous adipose tissues have similar trophic properties

Both adipose-derived MSCs expressed at the same level the trophic factors bFGF, PDGF and HGF (Fig. 3a). Accordingly, no differences were observed in their potential to promote human fibroblast proliferation and migration (Fig. 3b, c and d).

MSCs derived from omental and subcutaneous adipose tissues have similar immunomodulatory properties

The gene expression level of IDO was significantly lower in omental MSCs than in subcutaneous cells (p < 0.05. Fig. 5a). The level of IL-10 did not differ significantly between them. Both adipose-derived MSCs prevent CD4+ T cell-proliferation at the same extent (Fig. 5b).

A major challenge associated with MSC-based therapies is the selection of the source [14]. Here we showed in young and healthy female dogs that, compared to subcutaneous adipose tissue, omental adipose tissue is a more suitable source of MSCs. Since it can be procured from donors with any body condition, its collection procedure is always feasible, its cell yield is high and the MSCs isolated from it have desirable differentiation and paracrine potentials.

The fact that omental but not subcutaneous adipose tissue was always procurable may be attributed to volume variability of fat depots as well as to the expertise of the professional that procures them. In a study with 1265 dogs, it was shown that the size of collected samples was determined by the location of the adipose tissue, being visceral samples bigger than subcutaneous ones [12]. This difference might be critical when donor should be an emaciated patient due to either a chronic or a nutritional disease.

We showed that omental adipose tissue yielded a higher number of viable MSCs per gram of tissue than subcutaneous adipose tissue. These results are in agreement with other studies showing the same differences in humans [14] and dogs [10, 15].

Consistent with the results reported for human MSCs, canine MSCs isolated either from omental or subcutaneous adipose tissue followed a lineal trend of proliferation up to passage 12 and senescence characteristics appeared at passage 10 [23, 30]. Thus, our data support a significant but limited expansion potential of canine MSCs. Hence, the feasibility to be procured and the cell richness of the sample take higher relevance in order to choose the best source of canine MSCs.

Vasculogenesis is a crucial step in the wound healing process [44, 45]. The formation of new blood vessels is necessary to sustain the newly formed granulation tissue and the survival of keratinocytes. In this study, we found that adipose tissue derived MSCs express VEGF and ANG1. Both stimulate endothelial cell proliferation, migration, and organization into tubules [46, 47]. Our functional study showed that MSCs either form omental or subcutaneous tissue secreted active factors that promote vasculogenesis.

Since in the functional assays we used human fibroblasts or human endothelial cells as target cells, our data prove than trophic and vasculogenic factors secreted by MSCs isolated from dog omental or subcutaneous adipose tissue overcome species-specificity barrier. In order to further characterize the products secreted by canine MSCs it would be relevant to perform the functional experiments using target cells from dogs and other species.

Much attention has been paid to the immunomodulatory properties of MSCs. Several studies have shown a paracrine suppressive effect on T cells, B cells, monocytes and macrophages [48–50]. We showed similar gene expression levels of IL-10 in MSCs from both sources studied. Though, the expression of IDO in MSCs derived from subcutaneous adipose tissue was 11-fold higher than in MSCs derived from omentum. IDO catalyzes the conversion of tryptophan to kynurenine and inhibits T cell proliferation due to tryptophan depletion [51]. Nevertheless, this is not the unique mechanism supporting the immunosuppresive potential of MSCs [52–55]. That should explain why, despite of the differences observed in IDO mRNA levels, in the functional assay MSCs from both sources inhibit at the same magnitude CD4+ T cell proliferation. Our results appear consistent with previous findings for human and canine MSCs [4, 56, 57].

Data here presented not only shall be useful for evidence based-selection of MSC source but also to expand the frontiers of the use of canine MSCs as they prove to produce active trophic, vasculogenic and immunomodulator soluble factors.

Compared to subcutaneous adipose tissue, omental adipose tissue is a more suitable source of MSCs in dogs. Since it can be procured from donors with any body condition, its collection procedure is always feasible, its cell yield is high and the MSCs isolated from it have desirable differentiation and paracrine potentials.

Acknowledgements

We thank the dog owners for their disposition to participate in this study. Also, we are grateful with the people that work at the Center of Regenerative Medicine, Facultad de Medicina Clinica Alemana - Universidad del Desarrollo for their technical assistance.

This study was presented in part as an abstract at the International Society for Stem Cell Research Annual Meeting, Stockholm, june 2015.

Funding

This study was supported by CONICYT Grant No. 21110863 and by CONICYT Doctoral Scholarship to FB.

Availability of data and materials

All data supporting our findings are included in the manuscript. If readers need additional information they will be provided by the corresponding author (francisca.bahamondes@gmail.com).

Authors’ contributions

FB designed the study, performed the experiments, analyzed data and wrote the manuscript. EF and GC participated in animal management and adipose tissue sampling. FBr performed RT-qPCR. PC designed the study, supervised all procedures, analyzed data and revised the manuscript versions. All authors read and approved the final manuscript.

Consent for publication

Ethics approval and consent to participate

All dog owners gave written informed consent before animals enter in the study.

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